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Summary: Phosphatidylinositol 4-kinase IIα (PI4KIIα) generates essential phospholipids and is a cargo for endosomal adaptor proteins. Activity-dependent bulk endocytosis (ADBE) is the dominant synaptic vesicle endocytosis mode during high neuronal activity and is sustained by glycogen synthase kinase 3β (GSK3β) activity. We reveal the GSK3β substrate PI4KIIα is essential for ADBE via its depletion in primary neuronal cultures. Kinase-dead PI4KIIα rescues ADBE in these neurons but not a phosphomimetic form mutated at the GSK3β site, Ser-47. Ser-47 phosphomimetic peptides inhibit ADBE in a dominant-negative manner, confirming that Ser-47 phosphorylation is essential for ADBE. Phosphomimetic PI4KIIα interacts with a specific cohort of presynaptic molecules, two of which, AGAP2 and CAMKV, are also essential for ADBE when depleted in neurons. Thus, PI4KIIα is a GSK3β-dependent interaction hub that silos essential ADBE molecules for liberation during neuronal activity.

Huntington's disease (HD) is caused by CAG repeat expansion within the HTT gene, with the dysfunction and eventual loss of striatal medium spiny neurons a notable feature. Since medium spiny neurons receive high amounts of synaptic input, we hypothesised that this vulnerability originates from an inability to sustain presynaptic performance during intense neuronal activity. To test this hypothesis, primary cultures of either hippocampal or striatal neurons were prepared from either wild-type mice or a knock-in HD mouse model which contains 140 poly-glutamine repeats in the huntingtin protein (httQ140/Q140). We identified a striatum-specific defect in synaptic vesicle (SV) endocytosis in httQ140/Q140 neurons that was only revealed during high frequency stimulation. This dysfunction was also present in neurons that were heterozygous for the mutant HTT allele. Depletion of endogenous huntingtin using hydrophobically-modified siRNA recapitulated this activity-dependent defect in wild-type neurons, whereas depletion of mutant huntingtin did not rescue the effect in httQ140/Q140 neurons. Importantly, this SV endocytosis defect was corrected by overexpression of wild-type huntingtin in homozygous httQ140/Q140 neurons. Therefore, we have identified an activity-dependent and striatum-specific signature of presynaptic dysfunction in neurons derived from pre-symptomatic HD mice, which is due to loss of wild-type huntingtin function. This presynaptic defect may render this specific neuronal subtype unable to operate efficiently during high frequency activity patterns, potentially resulting in dysfunctional neurotransmission, synapse failure and ultimately degeneration.

Down syndrome (DS) is the most common genetic cause of intellectual disability, and arises from trisomy of human chromosome 21. Accumulating evidence from studies of both DS patient tissue and mouse models has suggested that synaptic dysfunction is a key factor in the disorder. The presence of several genes within the DS trisomy that are either directly or indirectly linked to synaptic vesicle (SV) endocytosis suggested that presynaptic dysfunction could underlie some of these synaptic defects. Therefore we determined whether SV recycling was altered in neurons from the Ts65Dn mouse, the best characterised model of DS to date. We found that SV exocytosis, the size of the SV recycling pool, clathrin-mediated endocytosis, activity-dependent bulk endocytosis and SV generation from bulk endosomes were all unaffected by the presence of the Ts65Dn trisomy. These results were obtained using battery of complementary assays employing genetically-encoded fluorescent reporters of SV cargo trafficking, and fluorescent and morphological assays of fluid-phase uptake in primary neuronal culture. The absence of presynaptic dysfunction in central nerve terminals of the Ts65Dn mouse suggests that future research should focus on the established alterations in excitatory / inhibitory balance as a potential route for future pharmacotherapy.

Synaptobrevin II (sybII) is a vesicular soluble NSF attachment protein receptor (SNARE) protein that is essential for neurotransmitter release, and thus its correct trafficking to synaptic vesicles (SVs) is critical to render them fusion competent. The SV protein synaptophysin binds to sybII and facilitates its retrieval to SVs during endocytosis. Synaptophysin and sybII are the two most abundant proteins on SVs, being present in a 1:2 ratio. Synaptophysin and sybII are proposed to form a large multimeric complex, and the copy number of the proteins in this complex is also in a 1:2 ratio. We investigated the importance of this ratio between these proteins for the localisation and trafficking of sybII in central neurons. SybII was overexpressed in mouse hippocampal neurons at either 1.6 or 2.15-2.35-fold over endogenous protein levels, in the absence or presence of varying levels of synaptophysin. In the absence of exogenous synaptophysin, exogenous sybII was dispersed along the axon, trapped on the plasma membrane and retrieved slowly during endocytosis. Co-expression of exogenous synaptophysin rescued all of these defects. Importantly, the expression of synaptophysin at nerve terminals in a 1:2 ratio with sybII was sufficient to fully rescue normal sybII trafficking. These results demonstrate that the balance between synaptophysin and sybII levels is critical for the correct targeting of sybII to SVs and suggests that small alterations in synaptophysin levels might affect the localisation of sybII and subsequent presynaptic performance.